The goal of this grant proposal is to develop genetic tools that can be used to activate or inactivate G-protein coupled receptor (GPCR) signaling cascades in rigorously identified populations of astrocytes using approaches that can be readily used by the research community; we believe the availability of such tools will be transformational with respect to understanding the role of astrocytes in physiology, behavior, and mental illness. While astrocytes make up a large portion of the CNS and express a wide variety of GPCRs that are activated during neuronal activity, the role of astrocytic GPCRs is poorly understood. The lack of information in this area stems from our inability to selectively activate or inactivate astrocytic signaling cascades while measuring physiological and behavioral parameters. Importantly, the majority of therapeutic agents used clinically to treat mental illness effect GPCR signaling cascades. Recently, genetic tools have been developed that can be used to selectively activate or inactivate GPCR signaling cascades in specific cell types. One such pharmacogenetic approach utilizes a receptor family referred to collectively as DREADD receptors (for Designer Receptor Exclusively Activated by Designer Drug) to activate GPCR signaling cascades in specific cell types. DREADD receptors were prepared by directed molecular evolution of M3-ACh receptor DREADD receptors, fail to respond to any known endogenous GPCR ligand, and are activated by clozepine-N-oxide (CNO). Importantly, CNO crosses the blood-brain-barrier enabling, for the first time, the selective activation of GPRC signaling in specific populations of astrocytes via an intraperitoneal (ip) injection of CNO. We plan to use intersectional gene activation which requires two cell- type specific promoters to activate gene expression and greatly increases the cell specificity of gene expression. Intersectional gene activation will be used to express Gq-, Gs-, and Gi-DREADD receptors in rigorously defined populations of astrocytes. A concern with any pharmacological approach is whether the resulting phenotype (e.g., LTP, behavior, etc.) reflects the over stimulation of the system under study. We plan to develop genetic tools that can be used to inactivate GPCR signaling cascades in rigorously defined populations of astrocytes; these genetic tools will also take advantage of intersectional gene activation. The combination of being able to activate or inactivate GPCR signaling cascades in rigorously defined populations of astrocytes will provide a powerful approach to sorting out the role of astrocytes in physiology, behavior, and mental illness; distribution of these mouse models to the research community is likely to be transformational. The genetic tools developed in this proposal will be used to test the hypothesis that the activation of astrocytic GPCR signaling cascades modulates synaptic transmission; this hypothesis is fundamental to our understanding of the role of astrocytes in brain function and is currently extremely controversial within the research community.